Anti psrc y416

Human E183 CTL were resuspended at 10⁶ per ml and 100 μl of the cells was added to each well containing the lipid bilayer and the specific pMHC monomer. The CTL were stimulated for 5 min at 37 °C, and the reaction was stopped by adding 100 μl of 8% paraformaldehyde. The cells were fixed at room temperature for 12 min. The samples were permeabilized with 0.3% Triton-X 100 for 4 min at room temperature, then blocked with 10% normal goat serum for 1 h. To visualize pLck or pZap70, the sample was labeled with 1:100 of anti-pSrc (Y416) (Cat# 6943 T) or anti-pZap70 (pY319) (Cat# 2717S) (Cell Signaling Technology) for 1 h at room temperature, followed by incubation with Alexa Fluor 488F(ab’) 2 fragment of goat anti-rabbit IgG (H + L) at a working concentration of 5 μg ml⁻¹ (Invitrogen). TIRF microscopy was performed on an Olympus IX83 inverted microscope fitted with a four-laser TIRF module. Images were acquired using a 100×/1.49 NA oil-immersion lens. Fluorescence excited within the 100-nm evanescent field was recorded with a Hamamatsu ORCA Flash 4.0 camera.

Western blotting was performed as previously described [29 (link)], using the following antibodies: mouse anti-Cbl-b, mouse anti-c-Src, and rabbit anti-β-actin antibodies were obtained from Santa Cruz Biotechnology; rabbit anti-Akt, anti-p-Akt (Ser473), anti-ERK1/2, anti-p-ERK1/2 (Thr202/Tyr204) and anti-p-Src (Y416) antibodies were obtained from Cell Signaling Technology, mouse anti-RANK antibodies were obtained from R&D, USA. Proteins were visualized using the enhanced chemiluminescence reagent (SuperSignal Western Pico Chemiluminescent Substrate; Pierce, Rockford, IL, USA) and signals were quantitated using NIH Image J software.

Antibodies used were as follows: anti-Eps8, anti-paxillin, anti-GM130 and anti-p130Cas antibodies (BD Transduction Laboratories, NJ), anti-FAK, anti-pSrc Y416, anti-Src (clone 36D10), anti-LC3B, anti-GAPDH, anti-pPaxillin Y118 and anti-β-actin antibodies (Cell Signaling Technologies, Danvers, MA), as well as anti-FAK antibody conjugated to agarose (clone 4.47) (Millipore, Billerica, MA) and anti-LC3B for immunoprecipitations (MBL, Woburn, MA). The anti-TagCGYFP antibody was purchased from Evrogen (Cambridge, UK). TRITC–phalloidin was purchased from Life Technologies (Paisley, UK). Horseradish-peroxidase-conjugated secondary antibodies against rabbit or mouse IgG were purchased from Cell Signaling Technologies. Dasatinib was obtained from Bristol Myers Squibb (Princeton, NJ). The pEGFP-Eps8 1–821 and pEGFP-Eps8 1–535 constructs were a generous gift from Giorgio Scita (FIRC Institute of Molecular Oncology, Milan, Italy).

HEK293 cells transfected with BmOR3 in the absence or presence of βarrestin1/2 siRNA were starved for 12 h at 37 °C before stimulated with 1 μM bombykal for 15 min. The cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM NaF, 1% Triton, 2 mM EDTA, 1% NP-40, 1% protease and phosphatase inhibitor cocktail), and the supernatant was collected after centrifugation. Proteins were separated by PAGE and transferred to PVDF membrane. After blocking in PBS containing 5% bovine serum albumin and 0.1% Tween-20, the membrane was incubated with the primary antibody at 4 °C overnight, followed by incubation with HRP conjugate–secondary antibody at RT for an hour. The signals were detected with the ECL system (Thermo Fisher Scientific). Primary antibodies were as follows: anti-ERK (Santa Cruz, Cat. No. sc-154); anti-pERK (Proteintech, Cat. No. 15361-1-AP); anti-SRC (Cell Signaling Technology, Cat. No. 2109S); anti-pSRC(Y416) (Cell Signaling Technology, Cat. No. 2101S); anti-beta actin (ORIGENE, Cat. No. TA811000); anti-βarrestin1(Proteintech, Cat. No. 15361-1-AP); anti-βarrestin2 (Proteintech, Cat. No. 10171-1-AP).